SF 796 

.ri6 

Copy 1 



Issued April 7, 1911. 



U. S. DEPARTMENT OF AGRICULTURE, 

BUREAU OF ANIMAL INDUSTRY.— BULLETIN 136. 
A. D. iMELVIN, Chief of Bureau. 



THE DIAGNOSIS OF GLANDERS BY 
COMPLEMENT FIXATION. 



BY 



JOHN R. MOHLER, V. M. D., 

Chief of the Pathological Division, 

AND 

ADOLPH EICHHORN, D. V. S., 
Bacteriologist, Pathological Division. 



[Reprint, October, 1911, with slight revision.] 




WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 

1911. 




GIass_ S-E44i». 

Book \^y 



7 

Issued April 7, 1911. 

U. S. DEPARTMENT OF AGRICULTURE, , ^ 

BUREAU OF ANIMAL INDUSTRY.— Bulletin 136. / » ; 

A. D. MELVIN, Chief of Bureau. 



THE DIAGNOSIS OF GLANDERS BY 
COMPLEMENT FIXATION. 

BY 

JOHN R. MOHLER, V. M. D., 

'1 

Chief of the Pathological Division, 
AND 

ADOLPH EICHHORN, D. V. S., 
Bacteriologist, Pathological Division. 



[Reprint, October, 1911, with slight revision.] 



WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 

1911. 



M^3 



^ 



^^ 



^^ 



^^ 



THE BUREAU OF ANIMAL INDUSTRY. 



Chief: A. D. Melvin. 

Assistant Chief: A. M. Farrington. 

Chief Clerk: Charles C Carroll. 

Animal Hushandry Division: George M. Rommel, chief. 

Biochemic Division: M. Dorset, cliief. 

Dairy Division: B. H. Rawl, chief. 

Inspection Division: Rice P. Steddom, chief; R. A. Ramsay, Morris Wooden, 

and Albert E. Behnke, associate chiefs. 
Pathological Division: John R. Mohler, chief. 
Quarantine Division: Richard W. Hickman, chief. 
Zoological Division: B. H. Ransom, chief. 
Experiment Station: E. C. Schroeder, superintendent. 
Editor: James M. Pickens. 
2 



n, 0* ffi. 



LETTER OF TRANSMITTAL. 



U. S. Department of Agriculture, 

Bureau of Animal Industry, 

Washington, D. C, March 20, 1911. 

Sir: I have the honor to transmit herewith a paper entitled " The 
Diagnosis of Glanders b}^ Complement Fixation," by Drs. John R. 
Mohler and Adoph Eichhorn, of the Pathological Division of this 
bureau. 

Since the discovery of the glanders bacillus in 1883 many efforts 
have been made to find a reliable method of making an early diag- 
nosis of the disease. The mallein test and later the agglutination 
test have been and are at present in general use, but neither of these 
is sufficiently reliable to be entirely satisfactory. Schiitz and Schu- 
bert, German investigators, recently called attention to the value of 
complement fixation as affording a more reliable method of diag- 
nosing glanders, and within the past year this method has been 
carefully studied and tested in the Pathological Division of this 
bureau. 

It will be seen from the details given in the accompanying paper 
that the authors have found the complement fixation test to be highly 
reliable as a diagnostic agent for glanders, and they present a thor- 
ough exposition of the method resulting from their searching experi- 
ments, including practical tests in a recent outbreak of glanders at 
Washington, D. C. 

In view of the great economic and scientific importance of the 
subject, and as no work upon this new method has so far been pub- 
lished in the United States, I recommend the immediate publication 
of the paper in the bulletin series of this bureau, in order that the 
value of the method and the technique necessary for its application 
may be made more fully available in this country. 
Respectfully, 

A. D. Melvin, 

Chief of Bureau. 
Hon. James Wilson, 

Secretary of Agriculture. 



CONTENTS 



Page. 

Introduction 5 

Hemohsis 7 

Method of obtaining hemolytic amboceptor (rabbit serum ) 9 

Titration of hemolytic rabbit serum 12 

Method of obtaining complement (guinea-pig serum) 14 

Titration of complement 14 

Specific complement fixation (deviation) 16 

Method of obtaining serum of animals to be tested 18 

Inactivating the serum 19 

Preparation of the antigen (glanders bacilli extract ) 20 

Titration of the extract 21 

The complement-fixation test 23 

Application of the test 24 

Controls _ 25 

Interpreting results of tests 27 

Controlling glanders in an infected stable 29 

Results of practical tests with complement fixation 29 



ILLUSTRATIONS. 



Page. 

Plate I. Diagrammatic representation of complement fixation 8 

II. Titration of hemolytic amboceptor (rabbit serum) 12 

III. Titration of complement (guinea-pig serum) 16 

IV. Titration of antigen (glanders bacilli extract) 20 

V. Final test showing positive reaction to glanders 26 

4 



THE DIAGNOSIS OF GLANDERS BY COMPLEMENT 

FIXATION. 



INTRODUCTION. 

The early diagnosis of glanders constitutes one of the most im- 
portant and difficult tasks which confronts the veterinarian engaged 
in sanitary work. This of course does not apply to the clinical cases 
of glanders, as in such cases the diagnosis is usually made without 
much difficulty from the characteristic symptoms and lesions present. 
In those instances, however, where there are no positive indications 
of the disease, it is impossible to establish a diagnosis by j^hj'^sical 
examination, and only through the aid of some special diagnostic 
method or test can there be any hope of determining the presence or 
absence of the disease. Horses affected with occult or latent glanders, 
and in which the disease is not suspected, are undoubtedly great fac- 
tors in the propagation of the infection. Indeed, there are many 
glandered horses which do not show positive symptoms until the 
later stages of the disease. 

Since the discovery of the glanders bacillus in 1883 by Loeffler and 
Schiitz the diagnosis of glanders has been the subject of numerous 
investigations, and as a result great progress has been made in its 
determination. After the isolation of the infective agent of the 
disease the diagnosis was confined to the demonstration and culti- 
vation of the organism, or to the reproduction of the disease by inoc- 
ulations of exudates or parts of diseased organs from affected horses 
into susceptible animals. 

The first important step toward determining obscure and latent 
cases of glanders was made by the discovery of mallein. With the 
aid of this biological product of the Bacillus mallei a large propor- 
tion of latent and occult cases of glanders can be diagnosed, partic- 
ularly when such tests are made by efficient and experienced veteri- 
narians. There are, however, a considerable number of glanderous 
animals in which the mallein fails to give a typical reaction, and, on 
the contrary, a reaction may follow the injection of mallein in the 
absence of glanders. Thus mallein is not an entirely reliable diag- 
nostic agent for determining glanders, nor has it ever been considered 

5 



D DIAGNOSIS OF GLANDERS. 

as efficacious in the detection of this disease as tuberculin for the 
diagnosis of tuberculosis. 

With the application of the agglutination test for glanders it ap- 
peared that a more satisfactor}^ method had been found for the diag- 
nosis of all types of infection with this disease. It was first sug- 
gested by McFadyean in 1896, after this investigator had observed 
the value of Widal's typhoid-fever agglutination test, but was not 
generally adopted until the method was perfected by Schiitz and 
Meissner, whose interesting results were published in 1905. This test 
has since been extensively employed in jiractically every country 
where glanders exists, and therefore ample opportunity has been 
furnished for drawing conclusions relative to its diagnostic value. 

While there is no doubt that the agglutination test is of great value 
in all cases of recent infection, the blood in such cases possessing a 
very high agglutination power (1 to 1,000 and higher), nevertheless 
extensive experience has proved that horses affected with chronic 
glanders give occasionally a very low agglutination value, which in 
some cases is even lower than that of normal blood serum (1 to 400 
or even lower). From this condition it appears evident that in cer- 
tain cases of chronic glanders the disease can be determined only by 
repeated tests, and a diagnosis in such cases is only possible from the 
fluctuation of the agglutination value — either an increase or a de- 
crease — as it is a well-known fact that this value remains stationary 
in normal horses. 

Besides this difficulty, there should also be taken into consideration 
the fact that the blood of normal horses sometimes shows a high 
agglutination value (1 to 800 and higher), and that changes in the 
agglutination power have been observed even in animals free of 
glanders. Furthermore, repeated agglutination tests require con- 
siderable time, as at least two weeks should elapse between two tests. 
Therefore the agglutination test alone does not constitute an entirely 
satisfactory diagnostic method for glanders. However, as its great 
value has been proved beyond doubt in the early cases of infection, 
it may well be utilized as an adjunct to any other test which may 
be applied in connection with the diagnosis of suspected cases of the 
disease. 

Hutyra compared the agglutination test with the mallein test from 
the tables included in the works of Schiitz and Miessner and of 
Nevermann, and came to the conclusion that the application of the 
agglutination test alone has not decreased the number of faulty 
diagnoses. He believes that the principal difference in the results lies 
in the fact that a large number of horses which were classified as 
only suspicious by the mallein test are considered as actually in- 
fected by the agglutination test. 



HEMOLYSIS. 7 

In further efforts to find a method by which an early diagnosis 
of glanders could be made, various investigators directed their atten- 
tion to the precipitation reaction. This is based upon the fact that 
when blood serum comes in contact with a concentrated extract of 
glanders bacilli the precipitins or receptors, which are formed in the 
blood of infected animals from the time the infection first occurs, are 
bound to the bodies in the bacillarj^ extract, producing a precipita- 
tion which is manifested by cloudiness at the point of contact of the 
two fluids. This method of diagnosing glanders has recentl}^ been 
recommended by Pfeiler ^ in Germany and by Konew ^ in Russia, 
but it has not been applied extensively in practice. This is probably 
due to the fact that the reading of the reaction is in some cases diffi- 
cult, due to the indistinct ring which occasionally is formed at the 
line of contact between the precipitant and the serum. 

In 1909 Schiitz and Schubert ^ published the results of their im- 
portant work on the application of the method of complement fixa- 
tion for the diagnosis of glanders. And since their experiments 
were followed b}^ splendid results, exceeding by far the results ob- 
tained from either the mallein or the agglutination test, they recom- 
mended that this method of diagnosis in combination with the agglu- 
tination test be taken as the official test in Germany. This method, 
overcoming as it does the disadvantages of the mallein and aggluti- 
nation tests, constitutes without doubt the most reliable method for 
the diagnosis of glanders which we have at our command at the 
present time. The complement-fixation test is, in fact, the most 
definite method known for determining specific infections and is as 
nearly perfect as a biological test can be. It has only recently been 
introduced in veterinary science and the publications concerning it 
are at present limited exclusively to foreign periodicals. The prin- 
ciple of this test is presented in the phenomenon of hemolysis, which 
was first discovered and studied by Bordet and Gengou, and extended 
by Ehrlich, Morgenroth, and Sachs. 

HEMOLYSIS. 

It is a well-known fact that if red blood corpuscles of one animal 
are introduced into another of a different species the blood of the 
latter acquires the power to dissolve the blood corpuscles of the 

1 Pfeiler, Willy. Die Ermittelung der Rotzkrankheit durch die Prazipitationsmethode. 
Archiv fiir Wissenschaftliche und Praktische Tierheilkunde. Band 35, Heft 4/5, pp. 
323-337. June 24, 1909. 

- Konew, D. Prazipitationsreaktion als diagnostische Methode beim Rotz. Vorliiuflge 
Mitteilung. Centralblatt fiir Bakteriologie. Abt. 1, Orig., Band 55, Heft 3, pp. 251-253. 
July 9, 1910. 

3 Schiitz and Schubert. Die Ermittelung der Rotzkrankheit mit Hilfe der Komplement- 
ablenkungsmethode. Archiv fiir Wissenschaftliche und Praktische Tierheilkunde. Band 
35, Heft 1/2, pp. 44-83. 1909. 



8 DIAGNOSIS OF GLANDERS. 

foiTner when mixed with them in a reagent ghiss. This reaction is 
termed hemolysis, which means the dissokition of blood corpuscles, 
thereby setting the hemoglobin free in the medium in which the 
corpuscles are suspended. 

To illustrate this phenomenon, if a rabbit is injected intraperi- 
toneally, intravenously, or subcutaneously with washed red blood 
corpuscles of a sheep, the blood of the rabbit will develop antibodies 
which possess a dissolving action for the sheep blood corpuscles; 
that is, the rabbit blood will contain specific hemolysins. 

The acquired hemolytic property of the blood depends on two 
substances. One of these is present in the blood of every animal, 
and is known as the complement. It is thermo-labile, which means 
that it is rendered inactive after the blood or serum has been heated 
to 5G° C. for half an hour. The other body, which is formed as a 
result of the injection of blood corpuscles, is thermo-stabile ; that is, 
it resists heating even higher than 56° C.. and is know^n as immune 
body, fixative, sensitizer, or hemolytic amboceptor. The name am- 
boceptor is derived from the fact that it has an affinity on the one 
hand for the blood corpuscles of the species of animal with which 
the animal has been injected, and on the other for the complement, 
this union taking place only after the first-mentioned affinity has 
been satisfied. 

These two substances, together with the corpuscles to be dissolved, 
comprise the hemolytic system, and their combination leads to 
hemolysis. (See PI. I, A.) This means that an opaque suspension 
of blood corpuscles is rendered semitransparent or " laked." The 
hemolysis, strictly speaking, does not represent a complete solution, 
but only an action of the hemol3^sin on the stroma of the erythrocytes, 
which permits the escape of the hemoglobin of the red blood 
corpuscles. 

The injection of blood corpuscles of one animal into another of a 
different species gives rise to the development of antibodies which 
confer upon the blood serum the hemolytic action. This phenomenon 
is somewhat similar to the production of receptors in the formation 
of antitoxins which are thrown off, but these receptors alone are not 
able to dissolve the red blood corpuscles, requiring also the presence 
of a ferment. This ferment, however, is a. constant constituent of the 
blood and is known as the complement. 

That both of these substances are constantly present in the hemo- 
lytic serum can be demonstrated in the following manner: If the 
hemolytic serum is heated to 56° C. for half an hour, thereby de- 
stroying the complement, this serum will no longer possess a hemo- 
lytic action; that is, it will no longer dissolve red blood corpuscles. 
This heating of the serum is known as inactivation. On the other 
hand, if to such inactivated serum there be added fresh ' untreated 



(Jul. 136, Bureau of Animal Industry, U. S. Dept. of Agriculture. 



Plate I. 




A. fc vi:^ + XrS = 

fleJi/ocdce//. ceatorr/fMit/ (/„i^n .freMocJc^// (aTnl7^"Si Jlem^Uis^,// result. 



(Sheep) 



niJ nmiccepfiir 
(no/Kmo/L,3). 



B. 






xfac^rto. 



rNoloct^Ho/u^i^) 











S8 Hoije ■serum frymolement 

90 /i)!/f, .a/anders l-omplemenr. 



Jjocte'n'a. 

(Gc/fin</er:s 
/Ofitiyen). 




Socter/o/usis 



/Va/iemo/ysiji /n)i//resultr 



Diagrammatic Representation of Complement Fixation. 



A, Hemolytic system. 

B, Bacteriolytic system. 

C, Negative reaction with normal horse serum. 
X>, Positive reaction with glandered horse serum. 



1 „ 



METHOD OF OBTAINING THE HEMOLYTIC AMBOCEPTOR. 9 

serum, which in itself has no henK)l3'tic properties, hemolysis will 
result. Thus by the addition of this ''resh serum a reactivation is 
accomplished. This is explained by the fact that through the heat- 
ing of the serum one of the substances necessary for the hemolysis 
has been destroyed, which is the complement. After the complement 
has been destroyed by heating it can be replaced by the addition of 
any normal serum, because it is known that the complement is present 
in all blood. However, the guinea-pig serum appears to be the most 
satisfactory in the application of hemolysis, inasmuch as it is very 
rich in complement and only a very small quantity is required to be 
added to the inactivated hemol3'tic serum in order to produce 
hemolysis. 

Accordingly, the substances necessar}^ for hemolysis are (1) the 
hemolytic amboceptor, which is the serum of a rabbit that has been 
injected with washed sheep blood corpuscles, (2) the complement 
in the form of normal guinea-pig serum, and (3) washed red blood 
corpuscles of a sheep. In the jireparation of these difl'eren( substances 
it is necessary to fix standards of practical constancy by proceed- 
ing along definite lines in the following manner: 

METHOD OF 0BTAI^■I^■G THE HEMOLYTIC A^IBOCEPTOK (rABBIT SERUm). 

Strong, vigorous rabbits are selected, and tliey are injected in- 
traperitoneally with a suspension of washed red blood corpuscles 
from a sheep. Three injections are made at intervals of seven days 
with 7 c. c, 10 c. c, and 12 c. c. of these blood corpuscles suspended 
in like quantities of phj'siological salt solution.^ 

The sheep blood is obtained by bleeding a vigorous sheep from 
the jugular. The side of the neck is clipped and shaved, and the 
part over the jugular disinfected with 75 per cent alcohol. Then a 
sterilized small-calibered trocar is inserted into the jugular, and the 
blood is collected in a sterile bottle containing a few glass beads. 
After the desired ((uantity of blood is obtained i( is shaken for 10 
minutes in order to defibrinate it. After defibrination, it is filtered 
through a double layer of sterile gauze into the glass tube in which 
the washing is to take place. The glass tube containing the blood 
is then filled with salt solution and placed in a centrifuge wdiich has 
a speed of 2,500 to 3,000 revolutions per minute. After the red blood 
corpuscles are thrown down the clear fluid above the corpuscles is 
pipetted off. Then the blood corpuscles are again thoroughly mixed 
,t with salt solution in the proportion of 1 to 9, and the centrifugaliza- 
tion is repeated. This washing should be can-ied out three or even 
four times in order to eliminate all the serum adhering to the red 



1 T'nlpss otherwise stated, the term " salt solution " In this bulletin refers to an 0.85 por 
I cent solution of sodium chlorid. 

10040°— Bull. 136—11 2 



10 DIAGNOSIS OF GLANDERS. 

blood corpuscles. Such washed blood corpuscles can then be used for 
the injection of rabbits in the preparation of hemolytic amboceptors, 
as well as for the test pro^Dcr. 

The washing of the sheep blood corpuscles must be thoroughly 
carried out, inasmuch as the presence of even traces of serum adher- 
ing to the corpuscles may cause difficulty in obtaining satisfactory 
results. If rabbits were injected with red blood corpuscles contain- 
ing a small quantity of serum, the rabbits would develop, not only 
antibodies, or immune bodies, but also coagulins and anticomple- 
ments, and the presence of these substances would give rise to diffi- 
culties in demonstrating the presence or absence of a complete hemol- 
ysis. Furthermore, if blood corpuscles containing even traces of 
serum were used in the tests it might produce a fixation of the com- 
plement and thereby give rise to errors. Such errors would occur 
particularly if the hemolytic action of the rabbit serum was not 
very high. 

The washed blood corpuscles should be used for the injection of 
the rabbit on the day the blood is drawn. For testing purposes, 
however, it will keep for two or three days in the ice chest. 

The rabbits to be injected are shaved on the posterior part of the 
abdomen, and the skin is disinfected with 75 per cent alcohol. They 
are then held with the head down by an assistant in order to prevent 
the puncturing of the intestines. The blood corpuscles to be in- 
jected are mixed with an equal quantity of salt solution and heated 
in a water bath to body temperature. Intravenous injections have 
been recommended by some investigators, but it was found in our 
work that intraperitoneal injections gave very satisfactory results, 
and furthermore, there is very little danger of losing rabbits from 
various complications by this method. 

After three injections with the quantities and at the intervals 
stated above, a small amount of blood is taken from the rabbit on the 
fifth or sixth day after the last injection. This blood is then titrated 
in order to determine whether its hemolytic action is of sufficient 
strength for future work. If the blood serum is found to be of a 
sufficiently high titre, the rabbit may be either bled to death, or, 
what is far more satisfactory, about 15 c. c. of blood may be drawn 
from the veins of the ear. By the latter method the rabbit may be 
used continuousl}' for the production of hemolj^tic serum. If it is 
desired to obtain the blood by bleeding the rabbit to death, the 
animal is anesthetized by a mixture of chloroform and ether, the hair 
on the neck is shaved, the skin is disinfected, and an incision made 
on one side of the neck in order to sever both the jugular vein and 
the carotid artery. Should the flow of blood cease on this side, the 
other side may also be cut. The blood is collected in centrifuge 



METHOD OF OBTAINING THE HEMOLYTIC AMBOCEPTOR. 11 

tubes, and after the bleeding has been completed centrifugalization 
of the blood is accomplished and the supernatant serum drawn off 
with a pipette. 

Bleeding of the rabbit through the veins of the ear is best accom- 
plished in the following way: After washing the ear and closely 
clipping the hair over the veins on the outside of the ear, the skin 
is disinfected with 75 per cent alcohol. Then a pledget of cotton is 
soaked in hot water (about 45° or 50° C.) and wound around the 
base of the ear in order to produce a hyperemia of the blood vessels. 
When a sufficient dilation of the vessels is observed, the middle and 
posterior auricular veins are severed, and the blood is then collected 
in centrifuge tubes. If the blood ceases to flow, the cotton should be 
removed from the base of the ear, and after placing it in hot water 
it is again applied to the ear and the hy2)eremia is thereby reestab- 
lished. In case the coagulation of the blood has prevented its flow 
at the place where the veins were severed, the wound may be scraped 
with a knife, and usually the blood will commence to flow freely 
again. The collected blood is then treated in the same manner as 
described above — that is, centrifuged — and the supernatant serum 
pipetted off. 

Should this hemolytic rabbit serum be used before it is 3 days old, 
it must be inactivated by heating to 56° C. for one-half hour. After 
this time the hemolytic rabbit serum is preserved with one-half of 
1 per cent of carbolic acid — that is, to each 9 c. c. of serum 1 c. c. 
of a 5 per cent carbolic-acid solution is added. Serum preserved in 
this way ma}^ be kept for two or three months. However, it should be 
retitrated every two or three weeks, as occasionally the titre of the 
serum drops. Such carbolized hemolytic serum does not require 
inactivation. 

"\"\1ien preparing hemolytic serum it is best to start with several 
rabbits, as occasionally one may die as a result of anaphylaxis, and, 
again, some rabbits are not adapted for the production of hemolysins. 

The titre of the hemolytic serum from a rabbit does not remain 
stationary, but for two or three weeks after the last injection it 
gradually lowers until it reaches that of a normal animal. If, how- 
ever, such a rabbit is reinjected with washed corpuscles after several 
weeks, the hemolysins will again appear after a short time. Hemo- 
lysins are kept more or less in reserve in the cells of such an animal. 
The renewed injection acts as a stimulant, and these bodies are 
quickly thrown off into the blood, while in a normal animal they 
form slowly, first being formed in the cells. Thus, the advisability 
of keeping rabbits which have been bled from the veins of the ear 
for further production of hemolytic amboceptors can readily be seen. 



12 



DIAGNOSIS OF GLANDERS. 



TITRATION OF HEMOLYTIC RABBIT SERUM. 



The hemolytic rabbit serum is titrated in order to establish the 
smallest quantity of serum that will produce hemolysis in the pres- 
ence of a certain quantity of complement and the suspension of 
washed blood corpuscles of a sheep. The amount of hemolytic serum 
that will produce complete hemolysis in tAvo hours at 37° C. is an 
amboceptor unit. 

For the preliminary work of titration 10 test tubes are taken, and 
dilutions of the hemolytic serum are made with salt solution in 
proportions of 1 to 100, 200, 100, 500, etc., up to 4,000. These dilu- 
tions are made from basic dilutions of 1 to 100 and 1 to 1,000 as 
may be seen from Table 1. 

Table 1. — Titration of rabbit scrum {Jiemolytic amboceptor). 



Tul>e. 


NaCl 
solution.! 


Amboceptor. 


Comple- 
ment. 2 


Blood cor- 
puscles. 3 


Remarks. 


1 
2 
3 
4 
5 
fi 
7 
8 
9 
10 


c. c. 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 


1 0. c. of — 
1: 100 

1: 200 

1: 400 

1: 500 

1: 000 

1: 800 

1:1,000 

1:1, .500 

1:2,000 

1:4,000 


c. c. 
0.5 
.5 
.5 
.5 
.5 
.5 
.5 
.5 
.5 
.5 


c. c. 




11 


3.5 
3.0 
4.0 




.5 




Complement control (no hemolysisshould occur). 


12 


1: 100 


Amboceptor control (no hemolysisshould occur). 


13 






Salt-solution control (no hemolysisshould occur). 











1 0.85 per cent NaCl .solution. 

2 0.5 c. c. of a 10 per cent solution of the complement. 

35 per cent suspension of sheep-blood corpuscles washed in salt solution. 

From each batch of serum taken from the rabbit a basic dilution is 
made in the proportion of 1 to 100, 'JOO, -100, etc., up to 4,000. From 
these basic dilutions the third column in Table 1 is made, as indicated 
below : 



A dilution of — 

200 is m:ide by adding 1 c. c. of 1 to 

400 is luado by adding 1 c. c. of 1 to 

500 is made by adding 1 c. c. of 1 to 

600 is made by adding 1 c. c. of 1 to 

800 is made by adding 1 c. c. of 1 to 
1,000 is made by adding 1 c. c. of 1 to 
1,500 is made by adding 2 c. c. of 1 to 1,000 dilution to 1 c. c. NaCl solution. 
2,000 is made by adding 1 c. c. of 1 to 1,000 dilution to 1 c. c. NaCl solution. 
4.000 is made by adding 1 c. c. of 1 to 1,000 dilution to 3 c. c. NaCl solution. 

It will be observed that the last three are made from the 1 to 1,000 
dilution. 



100 dilution to 1 c. c. NaCl solution. 
100 dilution to 3 c. c. NaCl solution. 
100 dilution to 4 c. c. NaCl solution. 
100 dilution to 5 c. c. NaCl solution. 
100 dilution to 7 c. c. NaCl solution. 
100 dilution to 9 c. c. NaCl solution. 



BuL. 136, Bureau of An;mal Industry, U.S. Dept. of Agriculture 



Plate 




TITRATION OF HEMOLYTIC RABBIT SERUM. 13 

The titration proper is then made in the following manner: Ten 
additional test tubes are each filled with 2.5 c. c. of salt solution, to 
which is then added the hemolytic serum (amboceptor) in quantities 
of 1 c. c. of the different dilutions to each tube. Thus, in the first 
tube we add 1 c. c. of the dilution of 1 to 100 ; in the next, 1 c. c. of 
the dilution of 1 to 200; and in the third, 1 c. c. of the dilution of 1 to 
400, etc. Afterwards the complement of the guinea-pig serum is 
added in quantities of 0.5 c. c. of a 10 per cent solution to each tube, 
and finally 1 c. c. of a 5 per cent suspension of sheep corpuscles in salt 
solution is placed in each tube. 

Besides these 10 tubes, there are also three control tubes, one to 
show that the complement alone (without the amboceptor) will not 
l^roduce hemolysis, the second that the amboceptor alone without the 
complement will not produce hemolysis, and the third that the salt 
solution alone will not produce hemolysis. Thus, in the first control 
tube we add 3.5 c. c. of salt solution, 0.5 c. c. complement, and 1 c. c. 
suspension of sheep corpuscles. In the second control tube we add 
3 c. c. salt solution, 1 c. c. amboceptor of the 1 to 100 dilution, and 
1 c. c. suspension of sheep corpuscles. In the third control tube we 
add 4 c. c. salt solution and 1 c. c. sheep corpuscles. It is advisable to 
place the tubes for the titration test in the lower row and the control 
tubes in the upper row of the test-tube rack. 

As can be seen from the various quantities added to the tubes, the 
final volume is alwaj's uniformly 5 c. c. Thus, the different amounts 
of the blood derivatives used are always made up to 5 c. c. by the 
addition of salt solution. 

After adding the substances in the test tubes in the order given, the 
tubes are well shaken, placed in a rack, and the rack put in an incu- 
bator at 37° C. for two hours. Then it is removed from the incubator 
and the results are read. (See PI. II.) 

The highest dilution in which complete hemolysis has taken place 
represents the titre of the hemolytic amboceptor. Thus, if complete 
hemolysis has taken place in the tubes where the dilution used in the 
rabbit serum Avas 1 to 2,000, the hemolytic amboceptor of that serum 
is represented by 2,000. This titre, however, is not used in the 
glanders test, but rather its double strength, which would be 1 to 1,000. 

The titre of the hemolj^tic amboceptor for use in glanders diagnosis 
should not be less than 1 to 1,000, and therefore if the rabbit serum 
should jDrove of a low^er titre it should not be used for this test. A 
low titre of the hemolytic amboceptor would disturb the results of 
the test, inasmuch as in such low dilutions too much serum would 
have to be used, and as the serum contains other substances in addi- 
tion to amboceptors it Avould have an influencing effect on the 
hemol3'sis. 



14 DIAGNOSIS OF GLANDERS. 

It is advisable to preserve the carbolized hemolytic amboceptor in 
small vials containing 1 to 2 c. c. of the rabbit serum, sealing them 
with paraffin. By this procedure the contents of a vial are used up 
more quickly and without frequent exposure. 

The titration test of the hemolytic amboceptor is given in consecu- 
tive order in Table 1, and the method of carrying out the tests relative 
to the addition of the different substances is likewise given in con- 
secutive order in all the tables. Thus the number of test tubes used is 
indicated, as well as the various substances and the quantities to be 
added, as they follow each other. It is our opinion that by this 
method the description of the tests can be followed without much 
difficulty, and by keeping these tables in the foreground the tests 
themselves may be applied readily, even by those who have had but 
little experience in this work. Of course accuracy in technique is the 
most important factor in the success of this line of serum diagnosis, 
and too much emphasis can not be laid upon this fact. 

METHOD OF OBTAINING COMPLEMENT ( GUINEA-PIG SERUM ). 

The complement represents the blood serum of a healthy guinea pig, 
and is obtained by bleeding the animal by severing the carotid and 
the jugular. 

The guinea pig is anesthetized with a mixture of chloroform and 
ether, and the neck is shaved and then disinfected with a 75 per cent 
solution of alcohol. The animal is held by an assistant Avith its head 
down, while the operator uses his left hand to pull taut the skin over 
the region of the throat and his right hand to make an incision on 
one side of the neck by which the carotid and the jugular are sev- 
ered. The centrifuge tube is immediately held under the opening, in 
order to collect all the blood. After the flow ceases, the same tech- 
nique is practiced on the other side of the neck. Care should be taken 
to avoid the cutting of the trachea. 

The blood thus collected, which usually amounts to 10 or 12 c. c, 
is placed in the ice chest, and after one or two hours the serum sepa- 
rates from the clot. The serum is then drawn off, and the rest is 
placed in a centrifugal machine, in order to obtain all the serum 
present in the clot. The guinea-pig serum should always be used 
fresh, and it is never advisable to use it after the second day, as the 
complement becomes considerably^ reduced upon standing. 

TITRATION OF COMPLEMKNT. 

It is desirable to titrate the complement of every guinea pig, as 
such practice will insure more accurate work and better results in 
the glanders test. By titration of the complement we aim to estab- 



METHOD OP OBTAINING COMPLEMENT. 15 

lish the complement unit which is the smallest quantity of comple- 
ment necessary to produce complete hemolysis in the presence of one 
amboceptor unit and a suspension of blood corpuscles of sheep. The 
smallest quantity is then taken as its test value. For the titration of 
the complement six test tubes are used, in addition to the three for 
controls. A 10 per cent basic dilution is made of the complement 
in the first test tul^e; that is, to 2.7 c. c. of salt solution 0.3 c. c. of 
complement is added. From this basic dilution the other dilutions 
are made by consecutively reducing the quantity of complement in 
the different test tubes. Thus, in the second test tube 0.5 c. c. of the 
10 per cent basic dilution is added, in the third 0.4 c. c, in the fourth 
0.3, and so on. Of course we add to each of these test tubes sufficient 
quantities of salt solution to make the required 3 c. c. Thus, into 
the second test tube we add 2.5 c. c, in the third 2.6 c. c, etc., of salt 
solution. After the complement has been added in quantities stated 
above, it is followed by the amboceptor. One cubic centimeter of 
the hemolytic amboceptor of which the titre has previously been de- 
termined is added, and finall}'^ 1 c. c. of a 5 per cent emulsion of blood 
corpuscles. The purpose of the controls is to establish in the first 
that the complement alone without the amboceptor does not produce 
hemolysis; in the second, that the amboceptor alone without the 
complement produces no hemolysis; and in the third, that the salt 
solution alone does not produce hemolysis. The first control tube 
contains 3.5 c. c. salt solution, 0.5 c. c. of the 10 per cent basic dilu- 
tion, and 1 c. c. of suspension of corpuscles; in the second control 3 
c. c. of salt solution, 1 c. c. of the amboceptor dilution, and 1 c. c. of 
suspension of blood corpuscles; in the third control, -1 c. c. of salt 
solution and 1 c. c. of suspension of blood corpuscles are used. 

After shaking all the tubes, the rack is placed in the incubator for 
two hours, and removed in order to read the results. (See PI. III.) 
The highest dilution of complement in the tube in which the hemoly- 
sis is complete indicates the titre of the complement. For instance, 
if hemolysis is complete in the tube where 0.3 c. c. of the 10 per cent 
basic dilution of the complement has been used, and hemolysis is in- 
complete in the tube in which 0.2 c. c. of a 10 per cent basic dilution 
has been used, then the titre of the complement is 0.03 c. c, inasmuch 
as we have started with a 10 per cent basic dilution. Thus, in the 
tests in this instance it would be necessary to use a 3 per cent com- 
plement solution. 

In Table 2, the titration of the complement is given as in Table 1 
for the hemolytic amboceptor, and this can be followed without much 
difficultv. Particular care, however, should be taken to use exact 
quantities as designated in the table. 



16 



DIAGNOSIS OF GLANDERS. 
Table 2. — Titration of complement. 



Tube. 


NaCl so- 
lution.' 


Comple- 
meiit.2 


Ambo- 
ceptor.' 


Blood 

cor- 

puscles.^ 


Remarks. 


1 


c. c. 

2.7 

2.5 
2.6 
2.7 
2.8 
2.9 


c. c. 
0.3 

6.5 

.4 
.3 

9 
•1 


c. c. 


c. c. 


Basic dilution of complement. 

Tubes 2 to (i are for establishing the smallest quan- 
tity of complement which produces complete 
hemolysis. This smallest quantity is then taken 
as its test value. For instance, if smallest quan- 
tity is 0.03, then to 97 c. c. NaCl solution 3 c. c. 
complement is added; if it is 0.02, only 2 c. c. Is 
added to 98 e. c. NaCl solution. 


2 
3 
4 
5 
6 




1 
i 
1 
1 
1 


7 


3.5 
3.0 
4.0 


.5 




1 
1 

1 


Complement control (no hemolysis should occur). 
Amboceptor control (no hemolysis should occur). 
Salt-solution control (no hemolysis should occur). 


8 


i 


9 











^ 0.85 per cent NaCl solution. 

-Guinea pig serum in diminishing quantities. 

"Of previously titrated hemolytic serum, double dissolving quantity. 

^ 5 per cent suspension of sheep blood corpuscles washed in salt solution. 

6 This amount is taken from the basic dilution in tube 1. 

The amboceptor should be inactivated when used fresh; that is, 
before it is three daj^s old. Otherwise it is carbolized with 10 per 
cent of 5 per cent carbolic-acid solution, and kept in ice box — in this 
case it is used without inactivation. 

Place tube rack in incubator for 2 hours and read results. 

This test should be made as a preliminary to every suspected 
glanders serum test, as it is always necessary to determine the smallest 
quantity of complement to be used for the final test. Of course, 
within 24 hours any number of tests can be made with the same com- 
plement dilutions. 

SPECIFIC COMPLEMENT FIXATION (DEVIATION). 

ComjDlement fixation is a biological reaction in which the phenome- 
non of hemolj'sis is emjjloyed as the fundamental princijDle. It is 
so called on account of the fact that the complement has been fixed 
by the combination of antigen with antibody and thus prevented 
from participating in the hemolytic process in which it is essential 
in order to have hemolysis. By this method even small quantities of 
amboceptors (antibodies) can be demonstrated in a serum. 

The presence of an infectious principle in the organism of an 
animal or a man has a stimulating effect on the production of anti- 
bodies (immune bodies). If a serum containing such immune bodies 
is inactivated and brought into contact with the antigen in the 
presence of complement, the complement Avill become firmly fixed 
by the combined immune body and antigen. (See PI. I, B.) Thus, 
anchoring takes place between the antigen and the antibody in which 
the complement becomes fixed. This anchoring is thoroughly estab- 
lished when the mixture is placed in an incubator for one hour. 
The addition of the hemolytic amboceptor and blood corpuscles to 



KL Industry. U. S. Dept. of AoRiculturs 



Plate III. 




SPECIFIC COMPLEMENT FIXATION. 17 

such an anchored antigen and innnune body will have no effect. (See 
PI I, D.) Thus, no hemolysis will take place, inasmuch as the com- 
plement has been fixed by the immune body and the antigen, thereby 
leaving the hemolytic system incomplete. On the other hand, if the 
inactivated serum contains no immune bodies, there would be no 
substance in the serum to anchor the antigen. As a result, therefore, 
no fixation of complement will occur, this being left free, and on 
addition of hemolytic amboceptor and blood corpuscles, hemolysis 
will now take place. (See PI. I, C.) Neither the antigen nor the 
antibody alone can fix the complement and thereb}^ influence hemo- 
lysis when the hemoh^tic amboceptor and blood corpuscles are added. 
HoAvever, in combination the fixation will invariably take place, and 
on the addition of the hemolytic amboceptor and blood corpuscles 
hemolysis will not be produced. 

Since the discover^' of this phenomenon it has been utilized exten- 
siveh'^ in serum diagnosis, but probably its greatest value has been 
obtained from the Wassermann reaction for the diagnosis of syphilis. 
It has also been employed in other diseases with more or less satis- 
faction, and its great field in bacteriological investigations has not 
yet been exhausted for the practical diagnosis and determination of 
immune bodies in serum. In veterinary practice complement fixa- 
tion is now gradually becoming used for the diagnosis of glanders. 
This method of diagnosing glanders has given the most favorable 
results in Germany, and constitutes at the present time the official 
test for Prussia and other parts of Germany. It has also been used 
in the diagnosis of other diseases of animals, but not with such suc- 
cess as in glanders. Particularly in tuberculosis the results were not 
uniform and otherwise not very promising. 

The presence of the specific immune bodies (bacteriolytic ambocep- 
tors) in the serum of glandered horses brings about the fixation of 
the complement wdien the antigen in the form of glanders bacilli 
extract is added to the hemolytic system. The serum of glandered 
horses, therefore, contains antibodies (immune bodies) against glan- 
ders bacilli, Avhich are specific only for the glanders bacilli and for 
no other infection. The complement fixation accordingly represents 
a specific test, as only in the presence of the glanderous immune 
bodies and glanderous antigen will the reaction take place. If, in- 
stead of the glanderous immune bodies, other antibodies of another 
infectious disease be present in the blood serum, they will exert no 
effect whatsoever on the glanderous antigen ; and, on the other hand, 
if serum containing glanderous immune bodies is brought in contact 
with an antigen of another infectious disease, it will also have no 
effect on the reaction. By this fixation of the complement the hemo- 
lytic system is left incomplete, and as a result no hemolysis will take 
place. This fixation of the complement by the antigen and immune 



18 DIAGNOSIS OF GLANDERS. 

bodies of glanders in the horse serum constitutes the diagnostic test 
for this disease. 

In the application of the test it is necessarj^ to have substances con- 
stituting the hemolytic system, which are the washed blood corpuscles 
of a sheep, the hemolytic amboceptor (rabbit serum), and complement 
(normal guinea pig serum). The quantity of the hemolytic ambo- 
ceptor to be used has been established by titration and described in a 
preceding part of this publication. In the test for glanders the 
double strength of the determined titre of the hemolytic amboceptor 
is used. A slight excess in the quantity of the amboceptor does not 
alter the outcome of the tests. 

On the other hand, the establishment of the smallest amount of 
complement which will i)roduce hemolysis in the presence of the 
hemolytic amboceptor and blood corpuscles is very essential, and 
accordingly this should be established by the previously described 
preliminary test, before tests for glanders are undertaken. By omit- 
ting this it is possible that an excessive amount of complement would 
be used which would in some cases affect the final results of the test. 
An oversupply of complement in the test may not only prove sufficient 
to be fixed by the immune bodies (antibodies) and antigen, but there 
might be also enough complement left to produce an incomplete or an 
almost complete hemolysis. Thus it is evident that it is of great 
importance to establish by such a preliminarv^ test (see Table 2) the 
exact quantity of complement to be used. Schiitz and Schubert found 
that the success of this method of diagnosing glanders depended 
greatly upon the proper quantity of complement used, and therefore 
the establisliment of this quantity should not be omitted. 

The red blood corpuscles of the hemolytic system always constitute 
a uniform quantity — that is, a 5 per cent suspension of the washed 
corpuscles in salt solution. As has been stated, this may be kept for 
testing purposes for two or even three days in the ice chest. The 
method of obtaining the corpuscles has been described in the early 
part of this work. 

In the complement-fixation test, there are also used, besides the 
hemolytic system, the serum of the horse to be examined and antigen. 

METHOD OF OBTAINING SERUM OF ANIMALS TO BE TESTED. 

The blood is drawn from the jugular vein of the suspected horse 
after a small area over the jugular has been clipped and disinfected 
with alcohol. The vein is dilated by pressure on the lower part of 
the neck, and the blood is drawn from the animal by the insertion of 
a trocar and cannula. It is recommended that the blood should be 
collected in uniform-sized tubes or bottles, which should be sterilized 
before using. A sufficient quantity of blood for testing purposes 



OBTAINING SERUM OP SUSPECTED HORSE. 19 

would be 50 to 100 c. c, and after allowing the blood serum in the 
tube to separate from the clot in a cool, dark place it is ready to be 
used for the test. If it is desired to forward the blood to a laboratory 
the tubes may be packed into separate containers or collectively in a 
box. Every tube should be labeled, and the number of the horse 
corresponding with the record number should be designated on the 
label. It is not absolutely essential to have clear serum, as in repeated 
tests carried out in this laboratory it was found that blood forwarded 
from Michigan to "Washington gave satisfactory reactions, although 
the serum was badly discolored as a result of disintegration of the 
blood corpuscles. If it is desired to preserve the serum, or if from 
some cause a test can not be applied during the first few days after 
the blood has been drawn, it should be preserved with a 0.5 per cent 
solution of carbolic acid. This percentage is best obtained by adding 

1 part of a 5 per cent carbolic-acid solution to 9 parts of the serum to 
be preserved. Such carbolized serum Avill respond to this test after 
several months. 

In cases where the mallein test has been used the blood of sus- 
pected horses to be examined for glanders by complement fixation 
should not be taken until from 7 to 10 days have elapsed after the 
last mallein test. This is necessary because of the possibility that 
the injected mallein may have exerted a stimulating effect on the cells 
with the production of immune bodies, and if serum is then taken 
for the test the results ma)^ lead to a faulty diagnosis. However, in 
suspected cases of glanders the blood of the animals may be drawn 
and forwarded for examination to a laboratory where the serum 
diagnosis of glanders is practiced, without the necessity of the appli- 
cation of the mallein test. 

INACTIVATING THE SERUM. 

Of the horse serum to be examined about 2 c. c. is drawn off and 
placed in a suitable tube or bottle in order to subject it to a tempera- 
ture of 58° C. in a water bath for one-half hour. This constitutes the 
inactivation of the serum ; that is, the complement which is present in 
the serum is destroyed by this heating. Such inactivated serum is 
ready for use in the testing, but should be used only on the da}^ of its 
inactivation. In case it becomes necessary to repeat the test, another 

2 c. c. of the sample should be inactivated. 

The method of inactivation referred to above applies only to 
horses. Miessner and Trapp found that serum of mules inactivated 
at 58° C. and 59° C. does not in all cases give satisfactor}^ results, 
as in many instances even the normal serum of these animals checks 
hemolysis. The numerous tests which were carried out in this labor- 
atory with serum of horses and mules proved that while the inactiva- 
tion of the horse serum at 58° C. and 59° C. always gave satisfactory 
results, the tests made with mule serum under the same conditions 
were far from uniform. 



20 DIAGNOSIS OF GLANDERS. 

Accordingly, it was deemed advisable to inactivate sera from mules 
at a hi<jher tein})ei'ature as well as by carbolization. Xormal mule 
serum was subjected to various degrees of temperature ranging from 
58° C. to G2° C. for tliree- fourths to one hour, and similar samples 
of serum were carbolized and inactivated at the same temperature 
as the noncarbolized serum. The results showed that carbolized mule 
serum inactivated at G0° C. gives the most uniform results in the com- 
plement-fixation test. The same serum if not carbolized showed 
checking of hemolysis at an inactivation recommended for horse 
serum. Inactivations at a higher degree of temperature are not 
advisable, inasmuch as at 62° C. a considerable percentage of sera 
w'ill coagulate. Therefore, in testing mule sera a carbolization with 
0.5 per cent carbolic acid followed by an inactivation at 00° C. for 
one hour is advisable. 

The antihemolytic action of the normal sera of mules is probably 
due to the presence of substances in the blood which exert a checking 
action on hemolysis, as glycogen, pepton, albumose. These undeter- 
mined substances act by using up the complement, having a greater 
affinity toward it than the hemolytic amboceptor has. 

PREPARATION OF THE ANTIGEN (GLANDERS BACILLI EXTRACT). 

The antigen represents a shake extract of glanders bacilli in salt 
solution. It is prepared as follows : From a stock culture of glanders 
bacilli subcultures are made on 2 per cent acid glycerin agar media* 
It is preferable to use Kolle flasks instead of tubes for the cultures, 
inasmuch as in such flasks the surface of the media is much larger 
and a greater quantity of bacilli can be obtained from them. After 
inoculating the media with glanders bacilli the flasks are placed in 
the incubator, and after 24 hours the condensation water in the cul- 
tures is allowed to run over the surface of the media. After another 
24 or 48 hours in the incubator the surface of the media contains 
usually a luxuriant growth of glanders bacilli, and it is then ready 
for washing. To each flask 20 to 40 c. c. of salt solution is added. If 
the cidtures have been grown in ordinary test tubes they are washed 
olf with 5 to 15 c. c. of salt solution. 

After the entire growth is washed off the surface of the medium 
the fluid is poured into sterile flasks and then heated to 60° C. for 
four hours in order to kill the glanders bacilli. After the heating 
of the bacilli the flasks are placed in a shaking apparatus and shaken 
for four days. 

After removal of the flasks the extract is placed in centrifuge tubes 
and centrifugalized for two hours in a centrifuge at a speed of 2,500 
to 3,000 revolutions per minute. Then to the clear liquid, which has 
been drawn off into suitable bottles, 10 per cent of a 5 per cent 
carbolic-acid solution is added, and the bottles are corked. This rep- 



3ui. 136, Bureau of Anim'.l Imdustrv, U. S. Dept. of AoRicuLTURt. 



Plate IV. 




TITRATION OF GLANDERS BACILLI EXTRACT. 21 

resents the extract or antigen to be used for the tests in a dihition 
which is established by titration. The extract usually keeps for two 
or three months, or sometimes even longer if kept in a dark, cool 
place. 

TITRATION OF THE EXTRACT. 

The titration of the extract is carried out in order to establish the 
quantity of the extract which no longer prevents hemolysis. First, 
dilutions with salt solution are made from the extract in proportions 
of 1 to 20, 30, 40, 50, and so on up to 200. These dilutions are made 
from a basic dilution of 1 to 10, as can be seen from Table 3. The 
titration proper is carried out as follows: Nine test tubes are used 
for the titration and three for controls. To each tube 1 c. c. of salt 
solution is added, then 1 c. c. of complement of the determined small- 
est quantity established according to the preliminary test (see Table 
2 ) . This is followed by the extract, each tube receiving 1 c. c. of the 
different dilutions prepared. Thus, in the first tube 1 c. c. of the 
dilution of 1 to 10 is added; in the second, 1 c. c. of the dilution of 
1 to 20; in the third, 1 c. c. of the dilution of 1 to 30, and so on. 
Then the rack is placed in the incubator for one hour. After it is 
taken out, 1 c. c. of a double quantit}^ of the previously titrated ambo- 
ceptor is added to each tube, and finally 1 c. c. of a 5 per cent sus- 
pension of washed sheep blood corpuscles. Of the three control 
tubes, the first serves for a control to show that the complement alone 
(without the amboceptor) does not produce hemolysis, the second to 
show that the anjboceptor alone does not produce hemolysis, and the 
third that the salt solution alone does not produce hemolysis. Thus, 
in the first control tube 3 c. c. of salt solution, 1 c. c. of complement, 
and 1 c. c. of blood corpuscles are added. The second tube contains 
3 c. c. of salt solution, 1 c. c. of amboceptor, and 1 c. c. of blood cor- 
puscles, while the third contains 4 c. c. of salt solution and 1 c. c. of 
blood corpuscles. This titration is similar to the titration of the 
hemolj'tic amboceptor. 

After shaking the test tubes, which should invariably follow all 
these additions, the rack is placed in the incubator for two hours. 
Then the results are read. The tube in which hemolysis is no longer 
prevented represents the titre of the extract. (See PI. IV.) In the 
glanders test, however, one-half of that quantity is used. Thus, if 
the titration should prove that the first tube which does not prevent 
hemolysis contains a dilution of 1 to 50, then a dilution of 1 to 100 
of the extract is used for the glanders tests. 

In Table 3 the titration of the glanders bacilli extract is given in 
consecutive order, and this may be followed in a similar manner as 
with the other tables. In this table the substances to be used are 
given consecutively, and likewise the exact quantities are indicated. 



22 



DIAGNOSIS OF GLANDERS. 
Table 3. — Titration of glandei's bacilli extract. 



Tube. 


NaCl 
solution. 1 


Comple- 
ment.2 


Extract. 3 




Ambo- 
ceptor.'' 


Blood 

cor- 

puscles.5 


Remarks. 


1 

2 
3 

4 
5 

6 
7 
8 
9 


c. c. 


c. c. 
1 
1 

1 
1 
1 
1 
1 
1 
1 


Ice. 

1 
1 
1 
1 
1 
1 
1 
1 
1 


of- 
10 
20 
30 

40 
50 
60 
80 
10< 
20 


X. 
3.. 


2 

03 

1 

.9 
1-1 

3 
O 


c. c. 


c. c. 

1 
1 
1 
1 
1 
1 
1 
1 
1 


One-half of the quantity of the ex- 
tract which no longer prevents 
hemolysis is the proper value to 

• be used in future final testing. 
Thus, if it should show by this 
test to be 1 to 50, then a dilution 
of 1 to 100 of the extract is used. 


10 


3 
3 

4 


1 






1 
1 
1 


Complement control (no hemolysis 


11 




1 


should occur). 
Amboceptor control (no hemolvsis 


12 






should occur). 
Salt solution control (no hemolysis 










should occur). 



1 0.85 per cent NaCl solution. 

2 The detennined smallest quantity established according to the preliminary test. 

3 A basic dilution is made from the extract in proportions of 1 to 10, 20, 30, etc., up to 200. From these 
basic dilutions the third column is made as indicated below. 

« Double the quantity previously determined by titration. 

6 5 per cent suspension of sheep-blood corpuscles washed in salt solution. 

The basic dilutions of the glanders bacilli extract are made as 
follows : 



A dilution of — 

1 to 20 is made by adding 1 c. 
1 to 30 is made by adding 1 c. 
1 to 40 is made by adding 1 c. 
1 to 50 is made by adding 1 c. 
1 to 60 is made by adding 1 c. 
1 to 80 is made by adding 1 c. 
1 to 100 is made by adding 1 c. 
1 to 200 is made by adding 1 c. 



10 dilution to 1 c. c. NaCl solution. 
10 dilution to 2 c. c. NaCl solution. 
10 dilution to 3 c. c. NaCl solution. 
10 dilution to 4 c. c. NaCl solution. 
10 dilution to 5 c. c. NaCl solution. 
10 dilution to l c. c. NaCl solution. 
10 dilution to 9 c. c. NaCl solution. 
c. of 1 to 100 dilution to 1 c. c. NaCl solution. 



c. of 1 to 
c. of 1 to 
c. of 1 to 
c. of 1 to 
c. of 1 to 
c. of 1 to 
c. of 1 to 



DIAGNOSIS OF GLANDERS. 23 

THE COMPLEMENT-FIXATION TEST. 

As previously stated, the serum of glandered horses contains 
immune bodies which develop during the course of the disease. The 
presence of these immune bodies in the serum is utilized in the devel- 
opment of the test. Naturally, the amount of these immune bodies 
in the affected horses is not uniform and undoubtedly depends on the 
extent of the infection present ; therefore it is advisable to use in the 
test such quantities of the serum as will prove sufficient for the reac- 
tion to take i^lace. 

The necessary quantity for the test has accordingly been established, 
through the painstaking experiments of Shiitz and Schubert, as 0.2 
and 0.1 c. c, placed in two different tubes. In some instances the tube 
containing 0.2 c. c. of the serum may show a fixation of the comple- 
ment, while the tube containing 0.1 c. c. of the same serum may show 
only a partial fixation or hemolysis. In the great majority of cases, 
however, the fixation is usually manifested in both tubes. 

The substances which are used in the fixation test have already been 
described in detail, their strength has been established by the titration 
of these different substances, and it need only be mentioned in passing 
that the best results will be obtained when the smallest quantities of 
substances permitted by the titration are used for the test; that is, 
the substances should have a high titre. If, for instance, the glanders 
extract should prove of a low titre, the albumins which are contained 
therein ma}' have an influencing effect upon the outcome of the test, 
inasmuch as it is known that albumins or cell extracts, if present in 
large quantities, will fix complements. Therefore it is always advis- 
able to use glanders extract of a high titre ; the quantity to be used 
should not be much above 0.01 c. c. 

Besides the centrifugal machine and an ordinary shaking appara- 
tus, only glassware is needed for the test. This consists principally 
of j)ipettes of various sizes, and there should always be a sufficient 
number on hand during the performance of the test. Those best 
adapted for the work are 1 c. c. pipettes, graduated into hundredths, 
and 10 c. c. pipettes, graduated into tenths. In addition, several 
pipettes of various sizes will be found useful for measuring the 
various substances to be diluted. Uniform-sized test tubes are 
advised and they should fit into test-tube racks. All the glassware 
should be cleaned and dry-sterilized before using. Only such a num- 
ber of tests as can be conveniently handled should be undertaken on 
a single dny. Finally, the incubator should contain sufficient space 
to hold the necessarj' number of racks during the test. With a good- 
sized incubator and the above-mentioned apparatus any State bacteri- 
ological laboratory could meet the demand for diagnoses from all 



24 DIAGNOSIS OF GLANDERS. 

the veterinarians within its borders, as from 80 to 100 tests may 
readily be made by this method in one day. 

APPLICATION OF THE TEST. 

Before undertaking the tests it is advisable to prepare the dilu- 
tions which will be used for the testing. The quantities of dilutions 
can be determined quite accurately, in accordance with the number of 
tests to be made. Thus the necessary quantity of a suspension of 
blood corpuscles is prepared, as Avell as the dilution of the necessary 
quantity of the amboceptor, complement, and glanders extract. The 
preparation of these dilutions is best accomplished in Erlenmeyer 
flasks, and the dilutions prepared should not be kept over one day. 

Four test tubes are used in testing the serum of each horse. For 
convenience in reading the results they are placed in a test-tube rack. 
The first test tube is marked with the number of the animal corre- 
sponding with that on the record for identification. Thus, for in- 
stance, should it be necessary to examine 20 horses, 80 tubes would 
be required for the tests. The first and third tubes are intended for 
the test proper, while the second and fourth tubes serve as controls 
for the horse serum to be examined. All the test tubes are first filled 
Avith the necessary quantity of salt solution, which will make up Avith 
the other substances used the required 5 c. c. Thus the first and third 
tubes each contain 1 c. c, of salt solution, while the second and fourth 
tubes contain 2 c. c. each. Then the suspected horse serum to be ex- 
amined, which has been previously inactivated by heating for one-half 
hour at 58° C, is added. In the first and second tubes 0.1 c. c. and in 
the third and fourth tubes 0.2 c. c. of this inactivated serum is added. 
This is followed by the addition of the glanders bacilli extract, which, 
however, is only introduced into the first and third tubes, 1 c. c. of 
the established dilution being used. The second and fourth tubes, 
therefore, do not contain glanders bacilli extract, as these tubes serve 
only as controls to show that the horse serum alone will not influence 
hemolysis. Thus in every test the amount of serum used in the tests 
proper (first and third tubes) is controlled by an equal amount of 
serum (second and fourth tubes). 

After the addition of the antigen (glanders extract), 1 c. c. of the 
complement is added to every tube in a dilution which has been 
established by the preliminary tests. (See Table 2.) 

At this stage of the testing each tube contains 3 c. c. of fluid. The 
rack containing the tubes is now shaken and placed in the incubator 
for one hour. This incubation is carried out in order to allow the 
anchoring of the antigen and the glanders immune body, in which 
the complement will become firmly fixed. Naturally, the anchoring 



THE COMPLEMENT-FIXATION TEST. 25 

would only take place provided the serum contains the glanders im- 
mune bodies; that is, when the horse is infected with glanders. (See 
PL I, D.) On the other hand, in the absence of a glanders infection 
the serum does not contain immune bodies, and therefore the antigen 
as well as the complement will remain free in the test tubes. (See 
PI. I, C.) 

After the time required for the incubation has elapsed the rack is 
removed and the other substances of the hemolytic system are 
added, namely, the hemolytic amboceptor and the blood-corpuscles 
suspension. 

To each test tube is added 1 c. c. of the previously titrated rabbit 
serum (hemolytic amboceptor), and finally 1 c. c. of a 5 per cent sus- 
pension of sheep blood corpuscles which have been previously washed 
with salt solution. The tubes, either individually, or collectively in 
the rack, should then be well shaken by hand, in order to mix the 
fluids thoroughly and to wash down any flecks of the solutions that 
might have adhered to the sides of the tubes. 

This practically concludes the test, and it is now only necessary to 
incubate the tubes for 10 hours, when the results may be read. 

CONTROLS. 

During the application of all tests control tubes are also used at 
the same time for comparative purposes. Four tubes are required 
for the control of the hemolytic system and two for the control of 
the glanders extract. The control of the hemolytic system is es- 
tablished by using in the first tube all substances constituting the 
hemolytic system, namely, the hemolytic amboceptor, the comple- 
ment, and the suspension of blood corpuscles. This tube after the 
conclusion of the test should show a complete hemolysis. In the 
second tube the hemolytic amboceptor is controlled without the com- 
plement, to prove that it does not produce hemolj^sis. The third tube 
serves as a control of the complement without the hemolytic system, 
also to prove that it does not produce hemolysis. The fourth tube is 
to show that the salt solution has no hemol3'tic action on the blood 
corpuscles. The quantities of the different substances used in the 
controls are the same as used in the regular tests for glanders. It is' 
necessary in the control tubes, as well as in all other tubes, to make 
up the quantity to 5 c. c. with salt solution. Thus, in the tube which 
serves for the control of the hemolytic system, 2 c. c. of salt solution 
is added, while in the control of the hemolytic amboceptor as well as 
in the control of the complement 3 c. c, and in the salt-solution con- 
trol 4 c. c, are added. The substances added to the control tubes are 



26 DIAGNOSIS OF GLANDERS. 

used in the same order as given in the test, and the incubation is also 
followed at the same time. 

As above stated, two test tubes are used for the control of the anti- 
gen (glanders bacilli extract), in addition to the four used for the 
control of the hemolytic system. These two tubes contain the hemo- 
lytic system, and to the first tube 1 c. c. of the extract used in the 
glanders test is added, while to the second tube is added 2 c. c. of the 
extract. The controls of the extract serve to prove that the extract 
used in the tests does not cause a fixation of the complement, and that 
even double the quantity of the extract which has been used in the 
glanders test does not fix the complement. In the extract controls 
we have in the first 1 c. c. of salt solution, while to the second tube 
no salt solution is added, inasmuch as the substances in this control 
make up the necessary 5 c. c. In the first tube, besides the 1 c. c. of 
salt solution, 1 c. c. of the extract is added, which is followed in con- 
secutive order with the substances of the hemolytic system, in quan- 
tities and dilutions used during the test proper, which have been 
previously indicated. 

The results in the control tubes after the conclusion of the tests 
should show the following: 

All the control tubes which contain, besides the hemolytic system, 
only serum of the suspected horse — that is, the second and fourth 
tubes — should show a complete or almost complete hemolysis. There 
should be no deposit of blood corpuscles on the bottom of these tubes, 
or only a very small quantity. The fluid should have the charac- 
teristic (laked) wine-red color. 

In the control tubes of the extract there should also be a complete 
or almost complete hemolysis. The same should be present in the 
control of the hemolytic system ; it should show a complete hemolysis. 
In the control tubes of the complement the hemolytic ambocepter, 
and the salt solution, there should be no hemolysis whatever ; that is, 
the blood corpulscles should be settled in the bottom of the tube, and 
the liquid above them should be water-clear. 

In Table 4 the final test for glanders is given in the order in which 
it should be undertaken. The number of test tubes to be used is 
indicated, and also the number of controls to be made. The sub- 
stances and quantities, as well as the dilutions, are also indicated in 
the explanatory remarks of the table. As already stated, six controls 
are sufficient for any number of tests undertaken on one day, if the 
same substances are used for all the tests. In making the comple- 
ment-fixation test it is always advisable to control the results further 
by using serum from an established case of glanders in one series of 
four tubes, and from a healthy horse in another similar series, for 
comparative purposes. 



Buu. 136, Bureau of Anim-l Industrv, U. S. Dept. of AamcuLTURt. 



Plate V. 




THE COMPLEMENT-FIXATION TEST. 
Table 4. — The final test for glanders. 



27 



i 

^"^^- solution.' 

1 


Suspected | Glanders 

horse ' bacilli 

serum. 2 1 extract.^ 

1 


Comple- 
ment.'' 




Ambo- 
ceptor.* 


Blood 

cor- 

puscles.6 


Remarks. 


1 

2 


c. c. 
1 

2 

1 
2 


c. c. 
0.1 

.1 

.2 

.2 


c. c. 
1 


c. c. 


o 

1 
1 

a 

3 
o 


c. c. 


c. c. 


Test tulie for the dose 
0.1 c. c. of suspected 
serum. 

Serum control for the 


3 

4 


1 


dose 0.1 c. c. of sus- 
pected serum. 

Test tube for the dose 
0.2 c. c. of suspected 
serum. 

Serum control for the 






dose 0.2 c. c. of sus- 
pected s^runi. 

Control for the quantity 
of extract used (hemo- 
lysis). 

Control for the double 


5 


1 




I 

2 








6 










quantity of extract, 
for greater accuracy 
(hemolysis). 

Control of the hemolytic 
system (hemolysis). 

Control of the comple- 
ment (no hemolysis). 

Control of amboceptor 
(no hemolysis). 


7 


2 
3 
3 

4 












8 






9 








10 



















(no hemolysis). 



' 0.85 per cent NaCl solution. 

2 Suspected horse serum to be inactivated for 30 minutes at 58° C. in water bath, in order to destroy the 
complement which is present in the serum of every horse. 
3 One-half of the quantity which does not prevent hemolysis and established by titration. (See Table 3.) 
* The detemiined smallest quantity established according to the preliminary test. (See Table 2.) 
6 Double the quantity previously determined by titration. (See Table 1.) 
«5 per cent suspension of washed sheep-blood corpuscles. 



INTERPRETING RESULTS OF TESTS. 

The results of the tests are manifested in most instances by a dis- 
tinct reaction which takes place in the test tubes. The indication of 
the results is only to be sought in the tubes numbered 1 and 3, which 
represent the actual tests for the presence or absence of glanders, inas- 
much as these tubes contain all substances required in the tests. 

We may thus obtain in these tubes either complete hemolysis, in- 
complete hemolysis, or no hemolysis whatsoever. The fixation of the 
complement is manifested by the absence of hemolj^sis, and therefore 
we have in the first and third tubes a settling of the blood corpuscles 
with the watery clear fluid above. Such a result indicates without 
doubt the presence of glanders. On the other hand, if the first and 
third tubes show complete hemolysis, the absence of glanders is 
thereby indicated. In the presence of glanders a fixation of the 
complement takes place, as a result of anchoring to the immune bodies 
and antigen (see PI. I, D), while in the absence of glanders, there 
being no immune bodies present, the complement is used up in the 
phenomenon of hemolysis (see PI. I, C). 

Then, again, we may have cases in which the fixation of the com- 
plement is incomplete ; that is, there is a settling of corpuscles in the 
bottom of the test tube, but the fluid shows traces of hemolysis. It 



28 DIAGNOSIS OF GLANDERS. 

does not show the characteristic saturated color of hemolysis, but 
only a tingeing with the hemoglobin. This is termed an almost com- 
plete fixation, and also indicates the presence of glanders. The pres- 
ence of the characteristic color in the fluid and a very slight deposit 
of corpuscles on the bottom should not be taken, as an indication of an 
infection, as such a condition may be brought on by various causes, and 
particularly so by the presence of nonspecific substances in the serum 
of the horse, which may cause a very slight checking of the hemolysis. 
But all cases where the results show a fixation of the complement (no 
hemolysis) or almost complete fixation (slight tingeing of the fluid 
above the settled blood corpuscles) indicate the presence of glanders. 

As a result of the different quantities of horse serum used in the 
two pairs of test tubes, it will be found occasionally that the test 
tube containing the larger quantity of serum (0.2 c. c. — third tube) 
shows a fixation of the complement, while the tube containing the 
smaller quantity of serum (0.1 c. c. — first tube) shows a partial fixa- 
tion of the complement. This also can be considered as a glanderous 
infection, as it shows that there are present immune bodies, but not 
to such an extent as to produce the fixation of the complement in the 
tube where the smaller quantity of the suspected horse serum has 
been used. In most instances of glanderous infections, however, it 
will be observed that the fixation of the complement is uniform and 
almost complete in both tubes. 

The reading should be made after the results in the test tube have 
been clearly established, and one should not be too hasty in drawing 
conclusions before the reaction in the test tubes is clear enough to 
establish the results of the test. 

In the illustrations in Plate V the results of the glanders tests are 
indicated by the presence of a positive reaction. In tubes 1 and 3 a 
complete fixation of the complement has taken place, there being no 
hemolysis, while tubes 2 and 4 represent the controls for the horse 
serum (no glanders extract being added), these tubes showing hemo- 
lysis. In the upper row of the rack the controls are indicated, and they 
show the results as they should be obtained in the course of testing. 

Should the horse prove glandered on the above test, the test may be 
repeated, and then serum quantities are used as follows: 0.1, 0.05, 
0.03, 0.02, and 0.01 c. c. One control is made of 0.1 c. c. as in tube 2 
of the test. By this method the smallest quantity of serum is estab- 
lished which will divert the complement, but it is not necessary as 
routine practice. 

Our own experience, as well as the work of Schiitz and Schubert, 
to whom we wish to extend our acknowledgments, shows that the results 
of the complement-fixation test should be interpreted as follows : 

1. Horses in which the serum produces a complete fixation of the 
complement in the quantities of 0.1 c. c. and 0.2 c. c. should be con- 
sidered as glandered. 



EESULTS OF PRACTICAL TESTS. 29 

2. Horses in which the serum gives a complete fixation in the 
quantity of 0.2 c. c. and an incomplete fixation in the quantity of 0.1 
c. c. should likewise be considered as glandered. 

3. Horses in which the serum produces an incomplete fixation of the 
complement in the quantities of 0.1 c. c. and 0.2 c. c. should also be 
considered as glandered. 

I 4. Horses in which the serum shows no fixation of the complement 
in either tube should be considered free of glanders. 

In order to reduce the possibility of error to a minimum the 
agglutination test may be applied to the latter cases, and if this 
shows a value of 1 to 1,000 or over, the animal should be considered 
as glandered. HoAvever, such cases are extremely rare. 

The agglutination test may be undertaken with the complement- 
fixation test without a great deal of difficulty, especially if the 
agglutination test is carried out in accordance with the method used 
at the present time in Germany, by which the results of the test can 
be obtained in about two hours. This consists of a modification of 
the agglutination test, in which by centrifugalization the agglutina- 
tion is hastened in the test tubes and the results can be read after 
the tubes have been placed in the incubator for two hours. 

The technique of this method of agglutination testing will not be 
explained here, but it is planned to describe it in a later publication 
on this subject. 

CONTROLLING GLANDERS IN AN INFECTED STABLE. 

Wlien glanders is discovered or suspected among the horses in a 
stable, the blood of all the horses in the infected stable should be 
drawn and tested in the manner previously described. All animals 
whose serum shows a complement fixation should be destroyed with- 
out further consideration. After the animals have been killed the 
stable should be thoroughly cleaned and disinfected. The animals 
which gave no reaction on the first test should be retested after three 
weeks, and should there be no indication of the disease in the second 
test the stable may be considered free from the infection. On the 
other hand, if on the second test one or more animals should give a 
reaction, the infected animals should be destroyed and all the re- 
maining horses should again be subjected to another retest after 
three weeks. Not until the last test proves that no animal is affected 
can the stable be considered as free from the infection. 

RESULTS OF PRACTICAL TESTS WITH COMPLEMENT FIXATION. 

In order to ascertain how long a time after infection with glan- 
ders elapses before the presence of the disease could be detected by 
the complement-fixation test, a horse was infected with glanders at 
the Bureau of Animal Industry Experiment Station by taking a 



30 DIAGNOSIS OF GIANDERS. 

loof)ful of glanders culture and rubbing it up and down the Schnei- 
derian membrane. Twenty-four hours after the infection the first 
sample of blood was taken from the jugular, and this was followed 
by taking samples daily for about a month. The horse was inocu- 
lated on January 17, and the blood which was taken on January 22 
gave a positive reaction to the complement-fixation test, thereby 
proving that five days after infection the presence of glanders could 
be ascertained by complement fixation. The animal at that time 
showed no clinical indication of the disease, but three days later 
(on the eighth day) the discharge from the nose appeared, and at 
the same time the characteristic swelling of the submaxillary lymph 
glands was noted. Repeated tests made with blood taken daily from 
the horse gave always the characteristic reaction. After four weeks 
from the time of the infection the reaction appeared to be less pro- 
nounced, this probably being due to the acuteness of the disease and 
the great amount of toxin which had been produced continuously in 
the animal. The testing of this horse was not continued after the 
disease had affected the animal to such an extent that it appeared 
dangerous to continue the work, owing to the extensive infection 
accompanied by a very profuse discharge. 

Since this method of diagnosis for glanders was inaugurated in this 
laboratory, large numbers of horses and mules have been examined 
in the District of Columbia, as well as animals from other parts of 
the United States. Many of the horses examined had clinical cases 
of glanders, while others were selected because they were reactors to 
the mallein test, some typically, and others atypically. A large pro- 
portion of the cases, hoAvever, were exposed or " contact " horses. 
From the number of tests already made — about 1,540 — the results indi- 
cate that in the complement fixation we have a method which in accu- 
racy is equal to the tuberculin test for the diagnosis of tuberculosis 
in cattle. The results of the tests thus far conducted show that at 
least 97 per cent of the cases of glanderous affections can be de- 
termined by the complement-fixation method. Furthermore, the 
affected horses in which a negative or atypical reaction occurs are as 
a rule either very old chronic cases of glanders, or those fresh cases 
of infection tested during the period of incubation. According to 
Hutyra and Marek,^ the diagnosis of glanders by the complement - 
fixation test has already given such accurate results that it may be 
considered as the best method for the determination of this disease 
at the present time. f 

A good opportunity was recently afforded to apply the test exten- 
sively in an outbreak of glanders which occurred during the month 
of January, 1911, in a fashionable boarding stable in the District of 



1 Spezielle Pathologic und Therapie der Haustiere. Third edition, Band 1, 1910, p. 717. 



EESULTS OF PEACTICAL TESTS. 31 

Columbia. At this stable two clinical cases of glanders were found, 
and the phj^sical examination of 60 horses in the stable showed three 
additional cases with not tyiDical but somewhat suspicious symptoms. 
The testing of all the horses in the stable w^as immediately under- 
taken, and as a result of the comf)lement-fixation tests 14 horses were 
found to be affected with glanders. Unfortunately it was impossible 
to obtain the consent of the proprietor to test the horses with mallein 
for a comparative study of these two methods of diagnosis. 

Careful post-mortem exaininations were made on all the reacting 
animals, and in every instance lesions of glanders were found in one 
or more organs or tissues. In some of the cases only a few character- 
istic pulmonary nodules were present, while in others the character- 
istic lesions of acute glanders of the lungs and liver were present. 
There was not a single case among these horses in which the diagnosis 
on post-mortem examination could have been doubted. 

After removing and killing the affected animals the stable was 
thoroughly cleaned and disinfected, and a second test was made with 
the blood of the remaining horses three weeks from the time the first 
test had been conducted. On the second test all the horses except one 
gave an absolutely negative reaction. This horse was destroyed at 
once, and the post-mortem examination revealed a caseated focus in 
the left bronchial lymph gland and about twenty small nodules of 
glanders in the lungs, all of which were evidently recent, having de- 
veloped without doubt during the interv'al between the tests, as a 
result of the previous heavy exposure. 

In all other cases where the test has been applied and in which 
careful post-mortem examinations were held after the destruction of 
the j)ositive cases the presence of glanders has been demonstrated. 
Of course not all the animals which gave a reaction Avere autopsied, 
inasmuch as in some instances the tests were made for State officials, 
the animals not being under the jurisdiction of the Department of 
Agriculture. Again, in other cases where the animals were destroyed, 
no data of the post-mortem examinations were furnished. 

Among the horses tested by the complement fixation there were 
a number of animals which gave an atypical reaction to the mallein 
test, but on the complement -fixation test they proved either absolutely 
positive or negative. Of these horses those which gave a positive 
reaction and were killed proved to be glandered. 

Table 5 shows the comparative results obtained with the mallein 
and complement-fixation tests : 



32 



DIAGNOSIS OF GL.ANDEES. 



^ 






Table 5. — Comparative results with maUein 


and 


eon 


tdcnicwt-fixation tests^ 




Positive to mallein test. 


Negative to mal- 
lein test. 


Atypical reaction to mallein 
test. 




Locality. 


To- 
tal. 


Response 
to com- 
plement- 
fixation 

test. 


Post-mor- 
tem. 


To- 
tal. 


Response 
to com- 
plement- 
fixation 
test. 


To- 
tal. 


Response 
to com- 
plement- 
fixation 
test. 


Post-mor- 
tem. 


Total 
ani- 
mals. 




Posi- 
tive. 


Nega- 
tive. 


Posi- 
tive. 


Nega- 
tive. 


Posi- 
tive. 


Nega- 
tive. 


Posi- 
tive. 


Nega- 
tive. 


Posi- 
tive. 


Nega- 
tive. 






9 
9 
6 
6 
7 
18 
1 
35 
19 
1 
6 
5 
2 
6 
5 
2 
1 


8 
5 
4 
3 

"'vi' 

1 
26 
19 

""b 
6 
2 
4 
2 
2 


1 
4 
2 
2 

7 

1 


3 












1 

""4 

1 

4 

6 

"""3' 

"""'i' 


"'h' 


1 




10 


Florida 




9 


8 


1 


6 
4 
3 

12 
6 
9 
8 

12 
1 
6 
2 
1 


24 


Illinois 










10 








11 

32 

3 


2 


9 

32 

3 


2 
12 

6 
6 
4 

t 
3 
2 


1 


...... 


19 


Maine 




7 


51 


Michigan 


27 












10 


Montana 


9 






17 
11 


4 
11 


13 






60 


North Dakota . . . 










42 


Pennsylvania 


1 
1 










2 


Nebraska 
















12 


Wvoming 
















7 


Washington 


















3 


California 


2 
8 


















6 


Texas 












4 
1 


'"'"i' 


4 






9 


Oregon 














3 


Canada 


1 
















1 


Miscellaneous . . . 










29 


20 


9 


20 




29 




















Total 


137 


103 


34 


3 


7 


83 


25 


58 


104 


44 


61 


22 


8 


325 



In addition to the above there have been 1,217 tests made upon 
horses with the complement-fixation method alone, and we have also 
tested by this method the blood of one lion, which gave a positive 
reaction, and the blood of one human suspected of having glanders, 
which proved negative. These two results were later substantiated. 

Of the above-mentioned 1,217 tests, 643 were conducted on horses 
at Washington, D. C, and of these 21 gave a positive reaction, all of 
which were subsequently confirmed on post-mortem. The remaining 
575 were from miscellaneous sources, 78 of which were positive, while 
497 were negative. 

In the 323 cases in Table 5 wherein the two tests are compared, 
the mallein test was confirmed by the complement-fixation test in 161 
cases and was not confirmed in 59 cases. There were 104 atypical 
reactions to mallein which were definitely diagnosed by complement 
fixation — 44 positive and 60 negative. Seven of the Maine reactors to 
mallein and three atypical reactors have been examined post-mortem 
without showing any evidence of glanders. 

In order to determine whether the fixation of the complement may 
be obtained occasionally in normal horses or in horses affected with 
various diseases other than glanders, a number of tests were made 
with the blood of apparently normal horses and, also, with horses 
suffering with various infectious and noninfectious diseases. One of 
these tests was made with the blood of a horse affected with swamp 
fever, in which the temperature registered 106.2° F. ; other tests were 
made with blood from horses affected with distemper, dourine, influ- 
enza, pneumonia, heaves, lameness, fistulous withers, forage poison- 
ing, etc. ; but in all these cases negative reactions were obtained. 

From these results the specificity of the test can be readily appre- 
ciated. Animals affected with glanders will give a positive reaction. 
Normal animals or those affected with diseases other than glanders 
will give no reaction. 

o 



